Recent molecular and biophysical studies of neuronal nicotinic acetylcholine receptors (nAChRs) have unveiled a rich diversity in both primary structure and function. While this diversity is certainly impressive, it is essential to determine why there so many distinct types of ACHR channels expressed by both central and peripheral neurons. The overall goal of this proposal is to test the hypothesis that a major physiological role of nAChR diversity is to allow selective modulation of particular nAChR subtypes in the control of synaptic transmission. This proposal converges on three aspects of nAChR channel modulation: the role of second messengers in nAChR modulation, the role of calcium in nAChR channel function and the physiological impact of nAChRs in the modulation of transmitter release at inter-neuronal synapses. We will examine the contribution of distinct nAChR channel subtypes and subunits to each of these modulatory phenomena in experiments addressing the following questions: 1: Do nAChR channel subtypes and subunits differ in their modulation by protein kinase C (activated by SP) and protein kinase A (by CGRP)? 2: Do nAChRs differ in their permeability to Ca and in their susceptibility to modulation by Ca? 3: What is the role of distinct nAChR subtypes and subunits in nicotine-mediated presynaptic facilitation? The proposed experiments employ both biophysical and molecular approaches. First, the modulation of native nAChRs will be examined by single channel recording. Then, the role of individual subunits in nAChR modulation will be assessed by selective subunit deletion with sequence specific antisense oligonucleotidls, The effects of subunit deletion on nAChR modulation will be assayed at the whole cell and single channel level. Finally, these experiments will be complemented by more traditional "combinatorial" approaches, examining the modulation of nAChRs expressed from mixtures of specific subunit cDNAs in Xenopus oocytes. Together these studies will determine both the channel subtype and nAChR subunit specificity of modulation and will examine receptor modulation from the molecular mechanisms to the physiological impact on transmitter release at an identified synapse. Detailed understanding of nAChRs is essential both because of the role of nAChRs in neural transmission and because of their status as prototypical ligand-gated channels. Thus, nAChRs at ganglionic synapses control numerous peripheral autonomic functions including the maintenance of vascular tone and hence, the regulation of blood pressure. Furthermore, the structural and functional diversity of nAChRs is common to all ligand-gated ion channels cloned to date, so that these studies will extend our understanding of the mechanisms underlying plasticity of transmitter-gated channels throughout the nervous system.